The primary objective of this project is to isolate the cDNA and gene for human histone H2A.X, and study the regulation of this protein and its mRNA in proliferating and quiescent cells. During the previous year, we isolated the cDNA for human H2A.X. This H2A related protein which had not been sequenced previously yielded some intriguing information concerning the relationship of the H2A histone species during evolution. Seven of the final carboxy-terminal amino-acids of human H2A.X are identical to the H2A from S. cere. There is homology with the carboxy-terminal sequences of at least one H2A in all studied lower eukaryotes. The mRNA is very unusual in that it contains stem-loop and U7 snRNP sequence motifs found in mRNAs from replication-dependent histone species, as well as polyA motifs. Two mRNAs exist in proliferating cultures, one terminating at the stem-loop motif and the other at the polyA motifs with a polyA tail. Later this year, a new postdoctoral fellow will being studies of the mechanism of this regulation. We have isolated a gene for H2A.X from human genomic DNA. Results of Southerns suggest that there is only one gene for this protein and that the H2A.X gene does not contain introns. These results suggest that the regulation of histone biosynthesis may be more complex than previously thought, with transcripts from the same mRNA being processed for histone synthesis in both proliferating and quiescent cells.Synthetic oligos may be useful in inhibiting this processing in a therapeutically useful manner.